ArticleActa parasitologica2026
Rapid and Simple Detection of Enterocytozoon Bieneusi Using Lateral Flow Assay Based on Recombinase Polymerase Amplification or Nested PCR Combined with CRISPR-Cas12a.
Article in Acta parasitologica, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
backgroundEnterocytozoon bieneusi is an obligate intracellular microsporidian pathogen. It primarily causes diarrhea and weight loss in infected humans and animals, resulting in substantial economic losses to the livestock industry. Therefore, establishing a highly sensitive and specific detection method for E. bieneusi is critical for its prevention and control.
methodscrRNA and recombinase polymerase amplification (RPA) primers were designed based on partial sequences of the 18 S ribosomal RNA gene and the internal transcribed spacer (ITS) of E. bieneusi. DNA extracted from fecal samples was amplified using RPA or nested polymerase chain reaction (PCR). PCR amplicons were treated with a Tris-saturated phenol-chloroform-isoamyl alcohol mixture to obtain purified target DNA, which was subsequently introduced into the CRISPR-Cas12a reaction system. Post-reaction detection was performed via qPCR instrumentation, fluorescence visualization, and lateral flow strip (LFS) assays. The operational parameters for E. bieneusi detection were subsequently optimized using RPA/CRISPR-Cas12a or nested PCR/CRISPR-Cas12a platforms. The aforementioned methodology was concurrently validated using 50 clinical specimens with known E. bieneusi infection status.
resultsThe limits of detection were 7.13 copies/µL for RPA/CRISPR-Cas12a and 2.35 × 10
conclusionsWe established the first integration of nested PCR with CRISPR-Cas12a for the detection of E. bieneusi. and were also the first to quantitatively explore the detection limit of Cas12a using non-amplified E. bieneusi DNA. This approach offers a rapid, specific, and highly sensitive diagnostic method. Furthermore, the wide selection of appropriate visualization methods facilitates adaptation to various laboratory conditions and sample template concentrations, enabling accurate result interpretation.
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